The formation of new blood vessels, called angiogenesis, is a process tightly regulated by a balance between pro- and anti-angiogenic factors and physiologically it is activated in wound healing, ovulation, and menstruation. However, it is also stimulated in pathologic conditions such as cancer, macular degeneration in the eyes, psoriasis, and diabetes. Since most tumors cannot grow beyond a few millimeters in the absence of new blood vessel formation, angiogenesis inhibitors have been explored as a drug target to be used in combination with several other cancer therapies. Several studies have explored the use of DNA vaccines, small tyrosine kinase inhibitors, siRNAs, ribozymes, antibodies, and receptor blocking agents aimed at better understanding the angiogenic mechanism and development of potential inhibitors. Specialized cancer treatments with anti-angiogenic agents approved by the FDA include the monoclonal antibody bevaciznmab (Avastin) and the small tyrosine kinase inhibitors SU11248 (sunitinib) and BAY 43-9006 (sorafenib). Although the clinical application of these drugs in cancer therapy are promising, drug resistance development and long-term side effects like hypertension and endothelium dysfunction remain a concern.
The pro-angiogenic factor VEGF is the most studied growth factor in this field due to its specificity and important role in the activation of all steps of angiogenesis in the endothelium vasculature. The splicing variant VEGF165 is the predominant form and had been shown to be up-regulated in the tumor microenvironment by hypoxia or activation of oncogenes like HER-2. VEGF is a glycoprotein and consists of an anti-parallel homodimer structure containing inter- and intra-disulfide bonds and it has been shown to bind to three receptor types: VEGFR-1 (fit-1), VEGFR-2 (flk-1 or KDR), and neurophilin-1 (NR-1). The VEGF:VEGFR-1 interaction exhibits high affinity although the role of VEGFR-1 is not fully understood. Research suggests its function in activated pathways in macrophages or endothelial progenitor cells (EPC). In the endothelial cells the majority of angiogenesis signaling (proliferation, migration and survival) proceeds via the interaction between VEGF and VEGFR-2.
The binding site of VEGF to its receptors has been characterized by crystal structure analysis as well as alanine scanning and reveals overlapping regions located at the poles in the homodimer. VEGF:VEGFR-2 interaction has been explored using antibodies that bind VEGF as well as the extra-cellular domain of VEGFR-2, identifying VEGF epitopes in the binding region that inhibit VEGF dependent pathways. The interaction between VEGF and VEGFR-2 has been identified and comprises residues at a loop region formed by the anti-parallel β-sheets β5-β6 in the VEGF protein.
Blockade of receptor-ligand interaction offers a validated and proven approach in drug development because receptor:ligand interaction is usually confined to a defined portion of the ligand and the receptor, and recent technologies have allowed the accurate identification of these binding regions. Peptidomimetics is the approach of reproducing the biological activity or binding properties in a smaller molecule, like peptides or modified peptides which were designed to mimic the desired region.
HER-2 (human epidermal growth factor receptor-2) is a member of the HER family of receptor tyrosine kinases and is overexpressed in about 30% of invasive breast cancers. HER-2 is essential for muscle spindle development and regulates the formation of neuromuscular synapses. High expression of HER-2 causes disruption of the HER network in tumor cells leading to increase survival of the tumors. HER-2 overexpression is not only limited to breast cancer and its amplification has been seen in subsets of gastric, endometrial, ovarian, lung, esophageal, and uterine cancers. The amount of HER-2 in cancer cells is much higher than in normal tissues and tumors with high levels of HER-2 expression always show intense immunohistochemical staining. This makes HER-2 a potential therapeutic target and also suggests that HER-2 targeted therapy will target most cancer cells in a given patient. The overexpression of HER-2 has also been shown in both the primary and metastatic sites which suggests that HER-2 therapy may have potential in all disease sites.
The upregulation of HER-2 is associated with increased expression of VEGF at both the RNA and protein level in human breast cancer cells and exposure of HER-2 positive cells to Trastuzumab significantly decreases VEGF expression. Shc, a downstream adaptor protein of the HER-2 signaling pathway has been identified as a critical switch for VEGF production showing that VEGF is a downstream target of the HER-2 signaling pathway. This shows that the effects of HER-2 on tumor cell behavior may be mediated in part through stimulation of angiogenesis. Angiogenesis is the growth of new blood vessels from pre-existing ones and contributes to the development of numerous types of tumors and their metastasis. VEGF, a well known pro-angiogenic factor is secreted by most tumor cells. VEGF stimulates angiogenesis by binding to its receptor VEGFR-2 which is expressed by both endothelial and tumor cells. Pertuzumab binds to the extracellular domain of HER-2 at sub-domain II thereby preventing receptor dimerization and signal transduction.
The oncoprotein HER-2 is also a ligandless member of the HER family of receptors and other members of this family are HER-1, HER-3 and HER-4. The absence of a HER-2 ligand makes it a preferred dimerization partner with other HER receptors. All members of the HER family have an extracellular domain, a single transmembrane domain and a cytoplasmic portion that contains a conserved tyrosine kinase domain flanked by a carboxyl terminal tail with autophosphorylation sites HER-2 is known to regulate the formation of neuromuscular synapses and also important in muscle spindle development. High levels of HER-2 causes dysregulation of the HER network resulting to transformation, tumorigenesis and resistance to cytotoxic effects of TNFα. HER-2 overexpressing breast cancers are biologically different from other breast cancers and are known to be resistant to hormonal agents, and have increased ability to metastasize to other organs of the body like the lung and brain. HER-2 upregulation is not only limited to breast cancers as its amplification has been reported in subsets of gastric, esophageal, ovarian, uterine, endometrial and lung cancers. HER-2 upregulation is always accompanied by VEGF upregulation both at the RNA and protein level and most drugs that target HER-2 are known to also down regulate VEGF expression. This implies that, the effects of HER-2 may partly be mediated by upregulation of VEGF. Immunization with both tumor and angiogenesis associated antigens showed synergistic effects. Tumor cells are known to up regulate the expression of VEGF and its receptors thereby stimulating angiogenesis.